Recently, genetic engineering techniques using small molecule double stranded RNA (dsRNA) have been reported.
RNA interference (RNAi) known as a representative technique thereof is a phenomenon in which mRNA is degraded nucleotide-sequence-specifically by dsRNA, and the expression of a polypeptide encoded by the mRNA is suppressed. The fact that genes are silenced by dsRNA was initiated by study on antisenses in nematodes. In 1998, Fire et al. found that dsRNA is a trigger of gene silencing, antisense RNA and sense RNA are not capable of suppressing the expression of genes, and dsRNA in which antisense and sense RNA strands are annealed can efficiently suppress the expression of genes. In the RNA interference, an enzyme called Dicer produces small interfering RNA (siRNA) from the dsRNA.
This biological phenomenon which takes place in the cytoplasm of a cell is widely used as a tool for molecular biological studies and for the purpose of drug discovery research that screens a target protein of a drug. Also, dsRNA artificially sent into cells can strictly suppress the copy number of a specific protein, even at a low concentration. Thus, drug development which attempts to utilize dsRNA as a nucleic acid pharmaceutical is being progressed.
In terms of suppression of interferon responses in a mammalian cell, dsRNA (siRNA) consisting of single stranded RNA with a length of conventionally 15 to 25 base pairs is selected as the synthetic dsRNA. In addition, dsRNA in which 2 to 4 bases are projected on the 3′ end side of each strand is frequently used. Furthermore, dsRNA, in which the projected end portion is thymine (T), particularly deoxythymidine (dT), is frequently used.
In order to develop dsRNAs as pharmaceuticals, the explanation of in vivo kinetics (distribution into tissues, metabolic rate, etc.) should be sought. However, detection and measurement technologies for small molecule RNA of a specific molecular species have not been yet established.
A method for detecting siRNA in which its projected end portion consists of DNA, that is, the 3′ end is DNA, has been developed, which includes adding polydeoxyguanosine (poly dG) to the 3′ end DNA of siRNA using a terminal deoxynucleotidyl transferase (TDT); synthesizing cDNA by reverse transcription reaction using a polydeoxycytidine (poly dC) primer that anneals to the produced poly dG portion; and subsequently performing PCR (e.g., Patent Document 1). In Patent Document 1, the poly dG is considered preferable as a nucleotide added to the 3′ end of siRNA, which can be distinguished from the poly A tail of endogenous mRNA. In order to detect a trace of siRNA from mRNA that is abundantly present in biological samples, such as cells, etc., it is known that the addition of polydeoxyadenosine (poly dA) to the 3′ end of siRNA should be avoided. Furthermore, in the same document, it is described that poly dG is preferable because, when poly dC or polydeoxythymidine (poly dT) is added to the 3′ end of siRNA, it is difficult to control the length of a tailed strand.    [Patent Document 1] International Publication No. WO 2009/098988